Amazingly, Escherichia coli degrades over 60% of the murein (peptidoglycan) from its sidewalls each generation and is able to reutilize the components. This process is known as recycling. The goat of this research is to identify and characterize all the enzymes used by E. coli to reutilize cell wall peptidoglycan components. Recycling involves over 10 enzymes that appear to serve no other purpose than to facilitate breakdown and reutilization of murein components. Several outstanding questions remain to be answered in order to have a full understanding of the process. The recycling pathway is required for beta-lactamase induction in many Gram negative organisms and, in addition, we have recently observed that mutations in certain recycling genes result in autoaggregation and biofilm formation. Thus the pathway is of interest in the health related areas of resistance to penicillins and biofilm formation. Specific aims: Aim #1: Determine the pathway for utilization of N-acetylglucosamine in the absence of the N-acetylglucosamine kinase. We have already achieved the original goal of Aim 1, namely, Identify the kinase gene and demonstrate its role in reutilization of N-acetylglucosamine. Aim #2: Determine how E. coli degrades anhydro-N-acetylmuramic acid. Our recent results indicate that a kinase and an etherase may be involved. Hence our current efforts are aimed at purifying these activities in order to identify the genes responsible for the activities. Aim #3: Determine the true inducer of beta-lactamase. Aim #4: Determine how expression of antigen 43 is related to the recycling of murein tripeptide. Antigen 43 causes autoaggregation which facilitates biofilm formation. Deletion of either murein peptide ligase (mpl) or murein peptide amidase A (mpaA) results in production of tripeptide and antigen 43 suggesting that accumulation of the murein tripeptide, L-Ala-gamma-D-Glu-meso-diaminopimelic acid, may be involved. [unreadable] [unreadable]